Pathogen detection using biosensors is commonly limited due to the need for both sensitivity and specificity in detecting targets within the mixed populations present in complex samples (Lim et al., 2005). The relatively high detection limits inherent in most systems are influenced by many factors, including low target concentrations, poor capture efficiencies, non-target detection (false positives/negatives) and interference by organic/inorganic constituents, all of which precludes adequate detection (Simpson-Stroot et al., 2008). Additionally, for those systems using immunochemistry for target capture or reporting (e.g., antibody sandwich assays), non-target cross-reactivity issues can be problematic, and adequately specific antibodies are often not available.
Some of these limitations may be overcome by combining two different molecular signature techniques, which would bestow added confidence for identifying the presence of targeted pathogens—in particular, the use of specifically-targeted fluorescently-labeled 16S rRNA gene oligonucleotide probes in conjunction with specifically-targeted antibodies. The dual level specificity (nucleic acid and protein) allows two levels of accuracy for detection and/or confirmation, as well as addressing cross-reactivity. For example, if the antibody available for a given target cross-reacts with other bacteria (related or otherwise), it could still be used for antibody capture-dependent biosensors, as long as a labeled nucleic acid specific probe was used to generate the signal (as opposed to a labeled detector antibody). This probe would only provide fluorescent signal to the appropriate target. Thus, the binding of unlabeled non-target cells to the antibodies becomes a null issue as no signal is generated.
The use of fluorescence in situ hybridization (FISH) to phylogenetically identify microorganisms without cultivation based on either 16S or 23S rRNA has become a mainstay of microbial ecology since its introduction (DeLong et al., 1989; Amann et al., 1990; Amann, 1995; Amann et al., 2001; Wagner et al., 2003; Daims et al., 2005). FISH has also been reported as a rapid method for pathogen identification in clinical and food settings (Kempf et al., 2000; Hartmann et al., 2005; Kempf et al., 2005; Peters et al., 2006; Wellinghausen et al., 2006; Bisha and Brehm-Stecher, 2009; Bisha and Brehm-Stecher, 2009). Although the predominant FISH application has been to study microbial community structure and spatial arrangements on solid supports, some applications have explored its usefulness for flow cytometric analyses (Amann et al., 1990; Wallner et al., 1993; Wallner et al., 1997; Fuchs et al., 1998; Hartmann et al., 2005; Kempf et al., 2005). This adaptation to a liquid phase processing for flow cytometry lends itself to facilitating biosensor applications, provided that conditions allowing for both probe and antibody recognition are met.
Traditionally, samples to be processed by FISH are fixed with paraformaldehyde (PFA) to stabilize and preserve them (Daims et al., 2005). PFA acts as a strengthening agent on the membranes of Gram-negative bacteria by cross-linking proteins to prevent lysis during hybridization, but can make Gram-positive bacteria highly resistant to probe uptake (Leong, 1994; Daims et al., 2005). Additionally, this cross-linking activity, while giving stability and excellent conditions for FISH, can severely inhibit any subsequent immunochemistry. To circumvent these problems, combined bacterial applications of FISH and immunostaining have typically involved extensive antibody incubation times or involved processing steps to overcome the fixative effects (Aβmus et al., 1997; Li et al., 1997; Ramage et al., 1998; Oerther et al., 1999), limiting their utility for rapid and high-throughput testing situations.
As formaldehyde and its derivatives are well known in the histopathology community to inhibit molecular analyses (e.g., immunostaining, immunohistochemistry or nucleic acid analysis), alternative tissue fixatives have been explored that are more conducive to downstream processing (Baumgärtner et al., 1988; Leong, 1994; Shibutani et al., 2000; Srinivasan et al., 2002; Cox et al., 2006). Methacarn solution has been found to be a non-cross-linking protein-precipitating fixative that does not appear to affect polynucleotide or protein analysis of fixed tissues and usually will give superior immunohistochemical results (Shibutani and Uneyama, 2002). This success with tissues suggests that methacarn solution may also be successful with fixation of bacterial cells and lend itself to facilitating the use of FISH in combination with immunolabeling for biosensor detection.
In the work described herein, we demonstrate a modified liquid FISH processing method used in conjunction with capture antibody targeted detection (CAT-FISH) to increase the specificity for biosensor assays. Detection of pathogens in pure cultures and seeded matrices was demonstrated on a cytometric bead array biosensor, using bead-bound capture antibodies with FISH labeled cells. Since the applications of both FISH and immunochemistry have been well established for use with complex sample matrices, this method should be easily adapted to other bacteria and biosensor platforms. The use of FISH in conjunction with antibody based biosensor assays for pathogen detection has not been previously reported.